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Adhesin Degradation Accelerates Delivery of Heat-labile Toxin
by Enterotoxigenic Escherichia coli
Roy et al, 2011
J Biological Chem 286(34):29771–29779
Tips for understanding the paper:
serine protease – protein which cleaves other proteins at a serine residue
type V (autotransporter) secretion system
a self-contained protein translocation system … a secretion system which transports
proteins across both the inner and outer membranes of the Gram (-) cell
The secreted protein has the following domains:
signal
peptide
passenger
domain
transmembrane
-barrel
The N-terminal signal peptide targets the protein to a transporter complex (Sec) in the
inner membrane which transports it across to the periplasm. In the process this signal
is cleaved from the protein.
The -barrel region inserts into the outer
membrane, forming a pore.
The passenger domain of the protein
slides through the pore to the outside of
the cell. It is released from the cell by
autocleavage at the junction between the
passenger domain and the -barrel
domain.
OM
periplasm
IM
Heat-labile toxin (LT)
an enterotoxin (protein) produced by ETEC (see MoMD page 201 and fig 16-2)
identical to cholera toxin
You should read MoMD page 132 (“structure-function organization of exotoxins”), page
133 (“cholera toxin”), and figure 9-3
Caco-2 cells
see Wikipedia article http://en.wikipedia.org/wiki/Caco-2
bacterial adhesin
see MoMD page 117 (“surface colonization”)
experimental nomenclature
“r” in front of the gene (as in rEatA) stands for “recombinant”
“p” after the name (as in rEatAp) indicates the passenger domain (a protein, not the gene)
rEtpA.myc.His
a recombinant protein, produced by fusing the EtpA gene to gene sequences encoding a
short domain from the myc gene, followed by a region encoding 6 consecutive histidines
The resulting fusion protein contains three domains:
a. EtpA
b. 10 amino acids of the myc protein sequence (a protein “tag)
c. 6 histidine residues (a protein “tag”)
This is useful because the protein can be purified using immobilized antibodies against the
myc tag or the polyhistidine tag. Alternatively the protein can be purified by
chromatography on a nickel column, to which the polyhistidines bind.
Figure 3d
This is a “far Western” blot, used to detect protein-protein interaction. Read the
description in the “Experimental procedures.” You may also read more about this method
by looking it up online.
If you do not understand the meaning of a word, LOOK IT UP!
If you do not understand a concept or technique, LOOK IT UP!
This is what scientists do. 